This invention is directed to the production of viral particles from retroviruses which are capable of transducing cells, for example, avian cells, including germ cells. In particular, replication deficient retroviral vector particles can be produced in accordance with the invention.
Replication deficient retroviruses are particularly useful in recombinant methodologies such as gene therapy procedures and in the production of transgenic animals, for example, transgenic avians. One particularly useful transgenic animal that can be produced using replication deficient retroviruses is the transgenic chicken.
The production of an avian egg begins with formation of a large yolk in the ovary of the hen with the unfertilized ovum formed on the yolk sac. After ovulation, the yolk and ovum pass into the infundibulum of the oviduct where it is fertilized, if sperm are present, and then moves into the magnum of the oviduct which is lined with tubular gland cells. These cells secrete the egg-white proteins, including ovalbumin, ovomucoid, ovoinhibitor, conalbumin, ovomucin and lysozyme, into the lumen of the magnum where they are deposited onto the avian embryo and yolk. Researchers have been successful in producing transgenic avians in which the tubular gland cells produce the exogenous protein and secrete it into the oviduct lumen along with the egg white protein for packaging into an egg. See, for example, Harvey et al, Nature Biotechnology (2002) vol 20, p 396-399, the disclosure of which is incorporated in its entirety herein by reference and U.S. Pat. No. 6,730,822, issued May 4, 2004, the disclosure of which is incorporated in its entirety herein by reference. This system offers outstanding potential as a protein bioreactor because of the high levels of protein production, the promise of proper folding and post-translation modification of the target protein, the ease of product recovery, and the shorter developmental period of chickens compared to other animal species used for heterologous gene expression. Significantly, retroviral production in transgenic animals such as chickens can be limited by the size of the insert allowed by the retrovirus. Typically, inserts contained in the retroviruses are limited to 2 to 3 kb. Production of integration competent virus is inhibited when insert size constraints are exceeded. Important methods used to produce transgenic avians such as chickens using retroviruses involve the introduction of replication deficient yet integration competent retroviral particles into embryonic cells.
Replication deficient retroviral vectors lack certain genes required for successful reproduction of the virus. Traditionally, to produce replication deficient retroviral vectors, nucleotide sequences encoding replication deficient retroviruses have been transfected into cells which stably produce the gene products required for replication of the replication deficient retrovirus. That is, certain nucleotide sequences required for the replication of the retrovirus are missing from the retrovirus but are present in the genome of the cell in which the viral particles are produced. One system that has been used to produce replication deficient ALV retroviruses involves the use of Senta cells and Isolde cells (Cosset et al (1993) Virology vol 195, p 385-395). The process involves first transfecting nucleotide sequences encoding the replication deficient retrovirus into the Senta cells which stably produce the gag, pol and envE proteins. Viral titer obtained in the Senta cells is typically <1000/ml. To increase the titer, the viral particles produced in the Senta cells are used to transduce Isolde cells which stably produce the gag, pol and envA proteins. The retrovirus produced in this manner can contain a neomycin resistance gene which allows for selection of Isolde clones or single colonies, some of which will produce particles at high titers >10,000/ml. In spite of the production of useable amount of viral particles being produced, the titers are still relatively low using this procedure. In addition, the process is laborious and time consuming, taking typically about three months.
What is needed are new methods of producing viral particles which require less time and less labor and allow for the insertion of larger nucleotide sequences in the recipient genome and result in high titers.